Elevated intraocular pressure (IOP) is one of the primary risk factors in the development of glaucoma. The trabecular meshwork (TM) is a critical tissue involved in the outflow of aqueous humor and regulation of IOP. Changes in the extracellular matrix (ECM) environment in the TM can alter the ability of aqueous humor to properly drain from the anterior chamber. The involvement of TGF? signaling pathways in the regulation of the ECM in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) and TGF? signaling crosstalk in the regulation of ECM and fibrogenesis in other tissues such as liver, kidney, lung, and skin. Here we propose that the TLR4 signaling pathway is also involved in the regulation of the ECM in the TM. Our hypothesis is endogenous pathological TLR4 ligands activate TLR4 and augment TGF?2 signaling sensitivity, leading to glaucomatous TM damage and elevated IOP. We will address this hypothesis with 3 specific aims. Specific Aim #1: will determine whether TGF?2-TLR4 signaling crosstalk regulates glaucomatous ECM production in primary TM cells in culture. We will utilize an endogenous TLR4 ligand (fibronectin extra domain A, FN-EDA), which is found in the glaucomatous TM, to activate the TLR4 signaling pathway in the presence and absence of TGF?2 and determine the effect on ECM components and TGF?2-TLR4 signaling molecules. Similarly, we will elucidate the effect of a selective TLR4 inhibitor (TAK-242) in the presence and absence of TGF?2. Specific Aim #2: will determine whether TGF?2-TLR4 signaling crosstalk regulates glaucomatous ECM production in the TM and leads to the development of ocular hypertension in mice. We will utilize our established inducible mouse model of ocular hypertension by intraocular injection of Ad5.hTGF?2 in Tlr4 mutant and Tlr4 wild-type mice. We will discover the role of the TLR4 activator, FN-EDA, on ocular hypertension utilizing FN-EDA null mice, and determine the signaling pathway involved in TGF?2-TLR4 crosstalk using conditional Bambi knockout mice, conditional Myd88 knockout mice, and in mice lacking the p50 subunit of NF?B utilizing our Ad5.hTGF?2 model system. Specific Aim #3: will determine whether TGF?2-TLR4 signaling crosstalk regulates glaucomatous ECM production in the TM and leads to the development of ocular hypertension in human donor eyes. We will determine the expression of TLR4 and downstream TGF?2-TLR4 signaling proteins in normal and glaucomatous human eye sections by immunohistochemistry. We will also elucidate the effect of a selective TLR4 inhibitor on TGF?2- induced ocular hypertension in a human anterior segment perfusion organ culture model. These studies aim to explore a novel pathway involved in the development of glaucomatous TM damage. The data will be invaluable to the field of glaucoma research and could provide new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.